Biotechnological production processes are increasingly employed to obtain biological compounds and fine chemicals. The progress of molecular biology techniques makes possible the mass production of a wide variety of biological compounds and fine chemicals such as proteins, antibodies, polysaccharides, antibiotics, amino acids, vitamins, alcohols, etc. Often, in order to increase the efficiency of the production, the gene(s) producing the desired end product(s) is (are) genetically modified and/or introduced into a heterologous organism. The production of the desired end product(s) then takes place in fermenters controlled by modern control techniques. The end products are either the cells themselves, extracted from the cells, or collected from the cell culture broth if the compounds are found therein (either by active or passive secretion processes or cell lysis).
It is common to see that the biomass produced at the end of the fermentation process contains the desired end product(s) but also active DNA molecules. Often, the active DNA molecules are recombinant DNAs. If left untreated, the recombinant DNA molecules could remain in the isolated end product and could also be released into the environment. There have been great concerns by the general public about the possible adverse effect of the remnant recombinant DNA materials in crops and food products on human health. In addition, concerns about the potential impact of the recombinant DNA on the environment have caused the authorities and institutions in most countries to issue statutory requirements and regulations calling for inactivation of the waste materials produced from the fermentation process before being released into the environment.
There are several ways to inactivate nucleic acids. For example, nucleic acids can be inactivated physically, and most commonly, by heat. U.S. Pat. No. 5,417,862 reports a method of inactivating the biological activity of DNA by heating the DNA to 60-100° C. in the presence of an acid. A similar method of degrading DNA by a combination of heat and acid is reported in U.S. Pat. No. 5,118,603. The heating methods require considerable amounts of energy, which add to the costs of the end product in large scale fermentation processes. Furthermore, depending on the nature of the end product, the harsh heat (and/or acidic) conditions could be detrimental to the integrity and activity of said product.
The nucleic acids can also be inactivated by other physical means. U.S. Pat. No. 6,165,711 reports a method using laser beams for disintegrating nucleic acids in a biologically active proteinaceous material. As another example, a method for inactivating microorganisms using high-intensity pulsed polychromatic light is reported in U.S. patent application Ser. No. 09/818,256, now abandoned. This method requires complicated light-generating devices to be used and constantly maintained, and thus is cost-intensive. The light beams may disintegrate not only nucleic acids, but also other biological substances or active compounds which will cause them to lose their desired properties.
The nucleic acids can also be inactivated chemically by acids or alkali. For example, in U.S. Pat. No. 7,435,567, a method using hypochlorous acids for induction of autodigestion of nucleic acids in a microorganism is reported. U.S. Pat. Nos. 5,417,862 and 5,118,603 described above use other types of acid for the degradation of nucleic acids. While these methods cause the disruption of nucleic acids, the acid and alkali conditions are severe. The severe conditions may cause the unwanted denaturation of certain biological compounds such as proteins. The amount of the acid and alkali used can be relatively large, which makes the method disadvantageous from the industrial production viewpoint as well.
Attempts have been made to inactivate nucleic acids of an organism enzymatically, such as by using a nuclease. U.S. patent application Ser. No. 13/127,825 reports a method for degrading host cell nucleic acids associated with vaccine production, where the method comprises a step of nucleic acid degradation by adding purified nuclease into the cell culture. In U.S. patent application Ser. No. 10/607,903, the construction of a transgenic bacterial strain expressing a heterologous nuclease gene in an amount effective to degrade nucleic acids is reported. While the method of adding nuclease in vitro such as the one reported in U.S. patent application Ser. No. 13/127,825 causes disruption of nucleic acids, the cost is high since large amounts of nuclease are required, and the efficiency of degradation is low because the cell wall of the host cell blocks the access to nucleic acids by the nuclease when added in vitro. The transgenic approach such as the one reported in U.S. patent application Ser. No. 10/607,903 allows the nuclease to be co-expressed with the host cell and thus gain access to the host nucleic acids in vivo. However, co-expressing a nuclease in a cell without any protection mechanism, such as methylation, will significantly stress the cell, resulting in weakened cell growth and reduced production of end product.
The problem underlying the present invention is therefore to provide a cost-effective way to degrade the nucleic acids of a host cell that produces biological compounds and fine chemicals, especially on an industrial production scale. A further problem underlying the present invention is to provide a method in which the nucleic acid degradation process is controllable and does not impede the production of the desired end product(s) in the host cell. The above problems are solved according to the invention by the subject matter of the present claims.